Partial Transfer of Citrus genome to Fortunella species using cell engineering techniques.

以圆叶金柑和森田脐橙橙子的胚性愈伤组织原生质体为材料，通过电融合获得了属间体细胞杂种。在获得的八种不同融合菌株中，六种表现出正常形态，而融合物是畸形的。所有再生植株的叶片形态均为中间型，叶片厚圆，具有多倍体的典型特征。通过流式细胞仪和根尖细胞染色体计数的倍性分析显示，这些植物的四倍体（2n=4x=36）。融合产物经RAPD和CAPS分析证实具有杂交性。此外，对叶绿体和线粒体DNA的CAPS分析表明，这些体细胞杂种在分析区域内含有圆叶柑的叶绿体和线粒体DNA，但没有发生重组;而将“将军”柑橘（C.retuculata布兰科，2n=2x=18）胚性愈伤组织原生质体与葡萄柚（C.paradisi Macf.，2n=2x=18）。利用RAPD和CAPS技术对再生植株的叶片性状和核基因组进行分析，证实了两个再生植株的杂种性。流式细胞仪和根尖细胞计数的两个杂种揭示了染色体数目为27。这些三倍体体细胞杂种转入田间后，形态正常，生长旺盛。这些体细胞杂种可能来源于愈伤组织中的单倍体细胞与叶肉细胞的融合细胞，但柑橘二倍体愈伤组织中单倍体细胞产生的机制尚不清楚。

Intergeneric somatic hybrids were produced between embryogenic callus-derived protoplasts of round kumquat (Fortunella japonica Swingle) and ‘Morita navel' orange (Citrus sinensis Osbeck) by electrofusion. Among the eight different fusion strains obtained, six showed nomal morphology, whereas the remainings were malformed. All of the regenerated plants were intermediate in leaf morphology and had thick and round leaves which are typical characteristics of polyploids. Ploidy analyses by flow cytometry and chromosome counting in root-tip cells revealed tetraploidy (2n=4x=36) of these plants. Hybridity of the fusion products were confirmed by RAPD and CAPS analyses. Furthermore, analyses of chloroplast(cp) and mitochondrial(mt) DNA by CAPS showed that these somatic hybrids contained cp and mtDNA of round kumquat without recombination in analyzed regions.While, somatic hybrids were produced between embryogenic callus protoplasts of ‘Shogun' mandarin (C.retuculata Blanco, 2n=2x=18) and leaf protoplasts of grapefruit (C.paradisi Macf., 2n=2x=18) by electrofusion. Hybridity of the two regenerated plants was confirmed by leaf characteristics and nuclear genome analysis using RAPD and CAPS. Flow cytometry and root-tip cell count of the two hybrids revealed a chromosome number of 27. These triploid somatic hybrids on transfer to field showed normal morphology and vigorous growth. These somatic hybrids might have derived from fused cells between haploid cell in callus cultures and mesophyll cell, although the mechanism involved in the occurrence of haploid cells from diploid callus cultures of citrus cultivars is still unclear.